The proposed research involves studies of how proteins promote the splicing of group I and group II introns. These introns use RNA-catalyzed splicing mechanisms, but require proteins for RNA folding and RNA struc- tural stabilization. For group I introns, we focus on the Neurospora crassa mitochondrial tyrosyl-tRNA syn- thetase (mt TyrRS; CYT-18 protein), which we identified genetically as functioning in splicing multiple mt group I introns. During the current grant period we obtained a co-crystal structure of a splicing active, C-terminally truncated CYT-18 protein with a bound group I intron RNA and an NMR structure of the flexibly attached C- terminal domain of a closely related, splicing-active fungal mt TyrRS. These structures provided insight into how CYT-18's N- and C-terminal domains function together in group I intron splicing, revealed structural adap- tations of both domains required for group I intron binding, and enabled us to trace the acquisition of this new RNA splicing activity to a fungal subphylum that includes important human pathogens. In the proposed re- search, we would use the structural information developed for CYT-18 to investigate key questions about how proteins promote RNA folding and evolve new RNA-binding functions. For group II introns, we have used the Lactococcus lactis Ll.LtrB intron as a model system for studying intron-encoded reverse transcriptases (RTs), which function in both RNA splicing and intron mobility. During the current grant period, we mapped RNA- protein interaction sites, obtained new mechanistic insights, identified highly active thermophilic group II in- trons, and developed new general methods for high-level expression of group II intron RTs. The latter open new approaches for proposed studies of the splicing function, structure, and evolution of group II intron RTs. In addition to proteins that stabilize the active RNA structure, the efficient splicing of mt group I and II intros in N. crassa and yeast requires the DEAD-box proteins CYT-19 and Mss116p, respectively. During the current grant period, we showed that these DEAD-box proteins function as general RNA chaperones that bind RNAs non- specifically and use their RNA-unwinding activity to resolve stable inactive structures (kinetic traps) that im- pede RNA folding. Additionally, we obtained X-ray crystal and SAXS solution structures of Mss116p that pro- vide the basis for proposed studies with potential to yield new fundamental insights into DEAD-box protein mechanisms. Finally, we developed a novel approach combining mass spectrometry and genetics to identify new host-encoded splicing factors for the yeast mtDNA group II introns aI5 and bI1. We propose to study the mechanism of action of these proteins and combine them with the DEAD-box protein Mss116p to reconstitute the complete physiological splicing apparatus for these important model introns. The proposed studies are in- tended to provide novel information about how proteins promote RNA folding and RNA catalysis, the evolution of introns and splicing mechanisms, and the function and evolution of aminoacyl-tRNA synthetases, RTs, and DEAD-box proteins, all relevant to human diseases.